Cyanobacteria are prokaryotic organisms that typically produce toxic metabolites called cyanotoxins. The most commonly encountered toxic compounds that are released by cyanobacteria are microcystins (MCs). MCs are a group of cyclic heptapeptides which have common amino acid sequences with a recurring motif of a 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) group present. At least 150 variants of MCs, such as microcystin-LR (MC-LR), have been identified from different cyanobacterial species. MC-LR is the most common cyanobacterial hepatotoxin, which is often released in water during harmful cyanobacterial blooms.
MCs cause concerns due to both their acute toxicity and chronic effects on humans and wildlife. Due to these concerns, the U.S. Environmental Protecting Agency (EPA) advises that the total concentration of MCs in drinking water should be ≤0.3 μg/L for children, and the state of Minnesota advises an even lower concentration (≤0.1 μg/L) of MC-LR in water. Similarly, the World Health Organization has set a provisional guideline limit for the maximum MC-LR concentration in drinking water of 1 ppb. As a result, robust and sensitive techniques are needed to detected sub-ppb concentrations of MCs in water samples.
MC-LR is one of the most hepatotoxic microcystins released by cyanobacteria. Besides ADDA, the MC-LR structure (FIG. 1) includes a leucine (L) at position 2 and an arginine (R) at position 4 (amino acids in these positions vary from one MC variant to another). In order to monitor the MC-LR concentration in water samples, robust techniques that can detect low concentration of MCs are needed. Determination of MCs is challenging because of a lack of MC standards, inconsistent recoveries during sample preparation, and matrix interferences. Several analytical techniques have been used to detect, identify, and quantify MCs. For example, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase inhibition assay have been used to quantify these toxins. These high-throughput methods typically report total concentration of multiple MC variants, and are therefore limited for the identification and quantification of specific MC variants.
Separations of MCs by HPLC, as well as their detection and quantification, have been demonstrated using UV and fluorescence detectors. LC-MS has been used for the analysis of MCs in water samples since it can provide reliable detection, differentiation, and quantitation of MC congeners. LC-MS and MS/MS detection and quantification of MCs have been reported using several different mass analyzers, such as quadrupole (Q), triple quadrupole (QqQ), ion trap, linear ion trap (LIT), time-of-flight (TOF), and Q-TOF. Most LC-MS/MS quantification methods have been performed using multiple reaction monitoring (MRM) mode of QqQ mass spectrometers.
While LC-MS and LC-MS/MS methods enable highly selective and sensitive quantitation of individual MC variants, they commonly need to utilize some form of preconcentration before the analysis. The preconcentration improves limits of detection (LODs) and limits of quantification (LOQs) of MCs, but it may lead to incomplete or irreproducible recovery of MCs before the LC-MS analysis. Low LOQs (50 ng/L) of MC-LR, MC-YR, and MC-RR have been achieved without preconcentration of the samples before the LC-MS/MS analysis using a LIT MS in full scan mode. Considering the importance of detection and quantification of toxins in drinking water, it is important to continue developing LC-MS and LC-MS/MS methods that can employ modern HPLCs and mass spectrometers for quantification of MCs. There is a need in the art for new and improved methods and systems for the detection and quantification of MCs, such as MC-LR.